Immunoassay General FAQs

Q: Do I have to use a wavelength correction?
A: Reading at dual wavelengths corrects for the optical density contributed by the plastic the plate wells are made of as well as lamp and instrument fluctuations. Our plates are chosen for their optical quality, therefore this correction is actually very small. If the dual wavelength feature is not available, data read at the recommended detection wavelength should be largely unaffected.

Q: Can I use a spectrophotometer to read my assay?
A: No, not unless the product specific instruction manual specifically gives you this option. A microtiter plate reader capable of detecting at the appropriate wavelength is needed to evaluate your data for most enzyme immunoassay and ELISA kits. The sample volume is too small to be reliably evaluated in a spectrophotometer.

Q: What species does your kit work with?
A: Kits where the molecule is conserved among all species (e.g. eicosanoids, steroids) are species independent, and may be used with any species. Kits that list a specific species in the name (e.g. human) are specific for that species. If the species is not specified in the manual, the product is likely species independent. The information may also be found on the product specific page on our website.

Q: How do I know if I need to dilute or extract my samples?
A: If your samples contain analyte levels within the dynamic range of the assay at the minimum recommended dilution for your sample type, you may dilute your samples and run directly in the kit. If your sample levels are below the limit of quantitation of the assay, extraction and concentration is necessary.  Some kits require extraction and this is specified in the product specific instruction manual.

Q: What is the minimum recommended dilution for samples? Can I dilute less?
A: This is the minimum dilution required to eliminate matrix interference in the assay. In general, this data is collected using a pool of samples from each matrix and evaluated using linearity, spike & recovery, and parallelism. A larger dilution may be necessary for your specific samples. You should perform your own validation experiments to determine the optimal dilution for your specific samples. However, we do not recommend diluting less without doing the tests. If you have low levels of analyte in your sample, extraction is a better option for you to return accurate sample values.

Q: Can you explain why I am seeing inconsistent standard curves run in culture media?
A: Often times, the serum supplemented into media can be problematic. In order to minimize this problem it is very important that the non-conditioned medium is at room temperature and is well-mixed before using to prepare your standard dilutions. Serum is more dense than water and tends to settle to the bottom of the vessel if not well mixed.

Q: Why are my ODs different than those shown in the instruction manual?
A: It is normal to see a small variation in OD values between experiments. OD values can be affected by small fluctuations in temperature. To assure that this is not a problem, do not leave the plate on a cold lab bench or under a vent for room temperature incubations. The relative level of signal can be improved by running the substrate incubation at 25 - 37°C.

Q: What is the best way to analyze my data? What is 4-PL curve-fitting?
A: For the most accurate results we recommend that 4 parameter logistic (4-PL) curve fitting software be used. The object of 4-PL is to find the line equation that best fits the data within a certain set of limitations. Even in immunometric assays, the dose response curve is very rarely a straight line. By choosing a better curve fit for your data, you will ultimately have more accurate returned sample values. If this software is unavailable the user will have to plot their data and calculate by hand. Most microplate readers come with software.